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I read an article recently, written by researcher from Department of Biochemistry, University of Washington, which stated that:

Similarly, success in de novo protein design bears on the question I get after every talk about the importance of the order of chain synthesis on the ribosome to protein folding; computational protein design calculations completely ignore the order of synthesis which hence cannot be critical to protein folding.

I was wondering, how could it be that the form that the protein is folded to, does not have anything to do with the amino-acid sequence that constitute this protein? What I mean, is in case I look at mirror image of a protein, would it fold the same? if I consider for example the sequencers: ser-gly-ala-glu-pro-asp and asp-pro-glu-ala-gly-ser, will they both fold the same? (I think those are d-protein and it's l-protein counterpart)

Can anyone provide evidence that this is, in fact, so. Or do I misunderstand the section quoted?

link to the paper: sci-hub.tw/10.1002/pro.3588

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    $\begingroup$ Welcome to SE Biology. I've tidied your question a little. Some tips on using the site. First, please finish the Tour. It would be useful also to read the Help on asking good questions. One way your question could be improved is to cite the article you quote from, preferably with a link, so we can double-check it. Second, I am not clear what you mean by "the order of amino acids". Do you mean that you would expect those synthesized first to fold first and independently of those later in the chain? $\endgroup$ – David Oct 6 at 10:01
  • $\begingroup$ @David thank you for your remarks. I've edited the question, I hope Iv'e managed to better explain what I mean. $\endgroup$ – E. Ginzburg Oct 6 at 11:16
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how could it be that the form that the protein is folded to, does not have anything to do with the amino-acid sequence that constitute this protein?

The quote by the researcher says that the form is unrelated to the direction of synthesis (N->C rather than C->N). It implies that all that matters is the amino-acid sequence that constitutes the protein, and that protein folding is driven by thermodynamic stability rather than by kinetics.

in case I look at mirror image of a protein, would it fold the same?

Yes, if you had an exact mirror image of a protein alone in solution it would adopt the same fold but mirrored. The N and C-temini would be on the same amino acids but all amino acids would be D rather than L chirality. This molecule would not generally be found in biology, since typically life uses L-amino acids. It would have different enzymatic behaviour on chiral molecules.

if I consider for example the sequences: ser-gly-ala-glu-pro-asp and asp-pro-glu-ala-gly-ser, will they both fold the same? (I think those are d-protein and it's l-protein counterpart)

These are not mirror images, they are entirely different molecules. They are not chiral counterparts (L and D). In general they would not fold to the same structure as the CO and N groups in the backbone are flipped, see more on reddit. However this is a topic of research and some reversable sequences have been found, see Zhang2016 and Mittl2000.

On a deeper level, the researcher's claim that synthesis direction (and hence synthesis broadly) is unimportant is not clear cut. For designed proteins it may be true but these are small and hyperstable. For larger proteins and protein complexes kinetics play more of a role, and chaperones may be used to protect a growing chain as it comes off the ribosome. See for example discussion in Sorokina2018 and Deane2007.

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  • $\begingroup$ Thank you for your reply. could you please further explain what's the difference between mirror image and reversed order of the protein? I thought that the two sequences I mentioned were mirrored. $\endgroup$ – E. Ginzburg Oct 8 at 4:36
  • $\begingroup$ Sure. A protein is just a molecule, and it is a molecule with chirality: en.wikipedia.org/wiki/Chirality_(chemistry). This means that the mirror image of the molecule is a different molecule, but the connection between everything is the same. You can also, separately, reverse a protein sequence, though calling this "mirroring" is a bit confusing as you are not reflecting anything in 3D space. In this case you get a molecule that actually has different connections: the termini are in different places and the C and N in the backbone are flipped when you reverse the sequence. $\endgroup$ – jgreener Oct 8 at 10:51
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I suspect the author meant something a little different. It's not just that it doesn't matter if a protein is synthesized from N- or C-terminus it's also not important for the end result of folding that the synthesis is gradual at all.

When designing proteins de novo they model the folding on a computer. The gradual addition of amino acid residues to the protein is not part of these models, the computations are made on the entire sequence at once. Still, when the designed proteins are synthesized on ribosomes they fold correctly. So the fact that the proteins are built step by step shouldn't influence how they are folded very much. As long as there is a sufficiently low free energy state it will be achieved.

Also, N-ser-gly-ala-glu-pro-asp-C and N-asp-pro-glu-ala-gly-ser-C are not exactly the mirrored images of each other. They just have terminal amino and carboxyl groups swapped. I imagine this can influence folding a bit.

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In addition to what is already said, I want to mention something more subtle. What other people have commented here is about an old 'dogma' due to Christian Anfinsen, which postulates that all that matters is the sequence of aminoacids + the microenvironment (pH, temperature, etc).

However, I think you might be confounding two other layers of biochemical complexity. One, L versus D aminoacids. If all the machinery of protein synthesis (including the ribosome and the messenger RNA!) was perfectly specular (i.e. D-amino acids and L- sugars, and the corresponding RNA sequence), everything else being the same, then the folding would be expected to be the same (although in practice, this is a new field of molecular engineering that is not that advanced). In fact, it is not clear why we have a particular chiral form of sugars and amino acids, and might as well be an accident of evolution.

The second thing that might be confounded in your question is the order in which a particular amino acid is added to a newly synthetized protein. Even though the previous answers seems to dismiss this as unimportant; it is in fact very important. Amino acids at the beginning of a protein chain are being folded as soon as they exit the ribosome. Thus this is quite an important process, as proteins aren't just 'waiting' to be completely synthetized for the folding to start: they are continously interacting with the environment (including chaperones!), so the folding does depend also in the order of synthesis.

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  • $\begingroup$ You're probably right that the order in which amino acids are added is important. It's just that the article quoted in the question seems to says that it isn't (in most cases). $\endgroup$ – Zlira Oct 6 at 17:32
  • $\begingroup$ Except aren’t there chaperones to PREVENT such initial inappropriate folding? $\endgroup$ – David Oct 6 at 18:57
  • $\begingroup$ The question is asking if the order of the protein affects the native structure of the protein. Amino acids do begin to interact and form structures as soon as they are incorporated into the growing peptide, but as the paper you referenced states "in no case has a protein domain been shown to be able to fold into its native structure while still inside the ribosome", this is because although the amino acids fold, they form kinetically trapped intermediates and not the native structure. The native state is generally formed once all amino acids are present, so it it independent of order. $\endgroup$ – pepsiandsoda Oct 6 at 19:09
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The quote is getting at protein structure is generally thermodynamically driven and not kinetically driven. The structures would be different if you took a mirror image of the sequence that you showed as the amino acid at the n and c terminus are different as is the orientation of all the other amino acids. However, if the ribosome were to be able to translate in the direction of c to n terminus rather than n to c terminus then the structure of the protein formed would be the same, that is what the quote is getting at. This is because like I say protein folding is typically thermodynamically driven, even though some amino acids come out of the ribosome first and form a structure (the kinetic structure as these amino acids came first) the structure with the lowest free energy is formed. This occurs because typically there is not a large energetic barrier for the protein to overcome to refold to find it's minimum free energy structure and so the thermal energy is sufficient to allow for the activation energy to be overcome and hence the process is thermodynamically driven to minimise the free energy of the protein.

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  • $\begingroup$ Thank you for your reply! I guess my problem is that I don't quite understand the difference between thermodynamically driven process and kinetically driven one. I'll make sure to read into that. $\endgroup$ – E. Ginzburg Oct 8 at 4:31

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